Methods of using anti-inflammatory compounds

ABSTRACT

The invention relates to the use of 3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one for preparing a medicament intended to prevent or treat inflammatory disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/833,022,filed on Aug. 2, 2007 now U.S. Pat. No. 7,662,967.

FIELD OF THE INVENTION

The invention relates to the use of3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one for preparing amedicament intended to prevent or treat inflammatory disorders.

BACKGROUND

Inflammation is an important component of physiological host defence.Increasingly, however, it is clear that temporally or spatiallyinappropriate inflammatory responses play a part in a wide range ofdiseases, including those with an obvious leukocyte component (such asautoimmune diseases, asthma or atherosclerosis) but also in diseasesthat have not traditionally been considered to involve leukocytes (suchas osteoporosis or Alzheimer's disease).

The chemokines are a large family of signalling molecules with homologyto interleukin-8, which have been implicated in regulating leukocytetrafficking both in physiological and pathological conditions. With morethan fifty ligands and twenty receptors involved in chemokinesignalling, the system has the requisite information density to addressleukocytes through the complex immune regulatory processes from the bonemarrow, to the periphery, then back through secondary lymphoid organs.However, this complexity of the chemokine system has at first hinderedpharmacological approaches to modulating inflammatory responses throughchemokine receptor blockade. It has proved difficult to determine whichchemokine receptor(s) should be inhibited to produce therapeutic benefitin a given inflammatory disease.

More recently, a family of agents which block signalling by a wide rangeof chemokines simultaneously has been described: Reckless et al.,Biochem J. (1999) 340:803-811. The first such agent, a peptide termed“Peptide 3”, was found to inhibit leukocyte migration induced by 5different chemokines, while leaving migration in response to otherchemoattractants (such as fMLP or TGF-beta) unaltered. This peptide, andits analogs such as NR58-3.14.3 (i.e.,c(DCys-DGln-DIle-DTrp-DLys-DGln-DLys-DPro-DAsp-DLeu-DCys)-NH₂), arecollectively termed “Broad Spectrum Chemokine Inhibitors” (BSCIs).Grainger et al., Biochem. Pharm. 65 (2003) 1027-1034 have subsequentlyshown BSCIs to have potentially useful anti-inflammatory activity in arange of animal models of diseases. Interestingly, simultaneous blockadeof multiple chemokines is not apparently associated with acute orchronic toxicity, suggesting this approach may be a useful strategy fordeveloping new anti-inflammatory medications with similar benefits tosteroids but with reduced side-effects.

However, peptides and peptoid derivatives such as NR58-3.14.3, may notbe optimal for use in vivo. They are quite expensive to synthesise andhave relatively unfavourable pharmacokinetic and pharmacodynamicproperties. For example, NR58-3.14.3 is not orally bioavailable and iscleared from blood plasma with a half-life period of less than 30minutes after intravenous injection.

Two parallel strategies have been adopted to identify novel preparationsthat retain the anti-inflammatory properties of peptide 3 andNR58-3.14.3, but have improved characteristics for use aspharmaceuticals. Firstly, a series of peptide analogs have beendeveloped, some of which have longer plasma half-lives than NR58-3.14.3and which are considerably cheaper to synthesise. Secondly, a structure:activity analysis of the peptides has been carried out to identifypharmacophores in order to propose small non-peptidic structures whichmight retain the beneficial properties of the original peptide.

This second approach yielded several structurally distinct series ofcompounds that retained the anti-inflammatory properties of thepeptides, including 16-amino and 16-aminoalkyl derivatives of thealkaloid yohimbine, as well as a range of N-substituted3-aminoglutarimides. (Reference: Fox et al., J Med Chem 45 (2002)360-370; WO 99/12968 and WO 00/42071). All of these compounds arebroad-spectrum chemokine inhibitors which retain selectivity overnon-chemokine chemoattractants, and a number of them have been shown toblock acute inflammation in vivo.

The most potent and selective of these compounds was(S)-3-(undec-10-enoyl)-aminoglutarimide (NR58,4), which inhibitedchemokine-induced migration in vitro with an ED₅₀ of 5 nM. However,further studies revealed that the aminoglutarimide ring was susceptibleto enzymatic ring opening in serum. Consequently, for some applications(for example, where the inflammation under treatment is chronic, such asin autoimmune diseases) these compounds may not have optimal properties,and a more stable compound with similar anti-inflammatory properties maybe superior.

As an approach to identifying such stable analogs, various derivativesof (S)-3-(undec-10-enoyl)-aminoglutarimide have been tested for theirstability in serum. One such derivative, the 6-deoxo analog(S)-3-(undec-10-enoyl)-tetrahydropyridin-2-one, is completely stable inhuman serum for at least 7 days at 37° C., but has considerably reducedpotency compared with the parental molecule.

One such family of stable, broad spectrum chemokine inhibitors (BSCIs)are the 3-amino caprolactams, with a seven-membered monolactam ring(see, for example, WO2005/053702 and WO2006/134385). However, furtheruseful anti-inflammatory compounds have also been generated from other3-aminolactams with different ring size (see for example WO2006/134385).Other modifications to the lactam ring, including introduction ofheteroatoms and bicyclolactam ring systems, also yield compounds withBSCI activity (see, for example, WO2006/018609 and WO2006/085096).

To date, the identification of broad classes of agents with BSCIactivity, and hence anti-inflammatory properties in vivo, has been basedon optimising potency of the BSCI activity. For example, previousdisclosures taught that introduction of 2,2-disubstitution (at thealpha- or key-carbon atom in the acyl side chain of acyl-3-aminolactams)leads to a considerable increase in potency as a BSCI, both in vitro andin vivo in models of acute inflammation, whether the 2,2-disubstitutedacyl group was open chain (see WO2005/053702), monocyclic (seeWO2006/134384) or polycyclic (see WO2006/016152).

However, potency of the desired pharmacological effect is only onefactor in determining whether an agent will make a useful humanpharmaceutical, albeit an important factor. In particular, thepharmacokinetics (or disposition of the agent within the body) exerts amajor effect on the utility of a particular agent. Pharmacokinetics(defined in its broadest sense, as the study of the effects of the bodyon the drug, in contrast to pharmacodynamics, which is the study of theeffects of the drug on the body) depends on a host of complexphysiological processes, including (but not limited to) absorption,plasma stability, volume of distribution (and in particular rate ofequilibration into ‘target’ tissues), metabolic transformation(including hepatic metabolism, such as cytochrome P450 isoenzymemediated oxidation, and phase II metabolism such as sulfation andglucuronidation, and extrahepatic metabolism, such as serum enzymicmodification), and excretion (such as renal clearance into urine andfecal elimination). These processes are often collectively referred toas the ‘ADME’ properties of the agent (ADME being an acronym forAbsorption, Distribution, Metabolism and Excretion).

Another important factor in determining the utility of an agent as ahuman pharmaceutical is safety. Many, if not all, compounds administeredelicit multiple effects on the body of which the desirablepharmacological effects are usually only a subset. The remaining effectsmay result in harm (toxic effects) or inconvenience (side-effects) tothe patient. The study of such properties of candidate pharmaceuticalagents is called toxicology or safety pharmacology. Unwanted effects canbe broadly classified into two types. Class effects are intimately tiedup with the desired pharmacological action, and (to a greater or lessextent) are an inevitable consequence of manipulating the chosenmolecular target. For example agents designed to prevent pathologicalinflammation will, to a degree, result in immunosuppression and anincreased risk of infection. This is because inflammatory tissue damageand infection are both inextricably linked to the degree of immunesystem activity. As a result, all molecules sharing the identicalpharmacological target will, to a greater or lesser extent, share classeffects. In contrast, compound effects are specifically associated witha particular compound structure, usually as a result of an (oftenunexpected) interaction with a target distinct from the intendedpharmacological target. In principle, it is possible to find anothermolecule with the same intended pharmacological effects but which iscompletely devoid of the compound-specific side-effects. Some compoundeffects are common (such as hERG interaction, which can result indangerous prolongation of the QT interval during heart pacing, resultingpotentially fatal cardiac arrythmias), while other compound effects maybe apparently unique to the particular compound.

Crucially, despite decades of pharmaceutical development experience,there is still no generally accepted method for predicting either theADME and pharmacokinetic properties of an agent, or its toxicology andsafety pharmacology. It is for this reason that explicit testing, firstusing in vitro assay systems (such as hERG-expressing cell lines), thenin animals and finally in phase I clinical trials in man, is aregulatory requirement worldwide for the development of a newpharmaceutical.

Methods have been described for predicting certain aspects of ADME frominspection of the molecular structure, and there can be little doubtthat experienced medicinal chemists can reliably rule out manystructures on purely theoretical grounds. An example of such a “rule ofthumb” (for it is no more dependable than that) would be Lipinsky's“Rules of Five”, based on the observation that most approvedpharmaceuticals meet certain criteria related to molecular weight,number of rotatable bonds and polarity. Similarly, it is generally wellknown that molecules with large, hydrophobic groups are more likely toshow an undesirable interaction with the hERG channel.

Such general guidelines, even when applied together, may be useful foreliminating unsuitable molecules but many very unsuitable molecules (forvarious reasons) would still slip though the net. Today, no-one wouldseriously countenance selecting a drug candidate from a class of activecompounds on purely theoretical grounds. As a result, the discovery of aparticular compound from within a class which has particularlyadvantageous ADME, pharmacokinetic, toxicological and safetypharmacological properties requires considerable practicalexperimentation among good candidates, and is a novel finding whichcould not be predicted even by those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

Here we describe the novel compound3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one (I), which hasnot previously been reported.

This compound is a specific member of the broad generic class of BSCIswhich have been described previously (for example, see WO2006/134385).However, we now demonstrate that while all the molecules of the classhave BSCI activity, compounds (I) has significantly superior propertiesfor use as a human pharmaceutical as a result of its combination ofADME, pharmacokinetic, toxicology and safety pharmacology properties,when compared experimentally to other members of the class.

The carbon atom at position 3 of the lactam ring is asymmetric andconsequently, the compounds according to the present invention have atleast two possible individual forms, that is, the “R” and “S”configurations. The present invention encompasses the two enantiomericforms and all combinations of these forms, including the racemic “RS”mixtures. With a view to simplicity, when no specific configuration isshown in the structural formula, it should be understood that the twoindividual enantiomeric forms and their mixtures are represented. Sinceenantiomeric inversion has no effect on the key ADME propertiesresponsible for the superiority of the compound (and additionally hasonly a small effect on the potency of the compound as a BSCI), bothenantiomeric forms, as well as their admixtures, represent specificexamples which are materially superior to the class in general.

Preferably, the compound of formula (I) according to this invention willbe the compound of formula (I′).

The compound (I′), having the (S)-configuration at the stereocentre, is5 to 25 fold more potent as a BSCI than the (R)-enantiomer.

Also provided are pharmaceutical compositions, comprising, as activeingredient, a compound of general formula (I) or (I′), or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient and/or carrier.

By pharmaceutically acceptable salt is meant in particular the additionsalts of inorganic acids such as hydrochloride, hydrobromide,hydroiodide, sulphate, phosphate, diphosphate and nitrate or of organicacids such as acetate, maleate, fumarate, tartrate, succinate, citrate,lactate, methanesulphonate, p-toluenesulphonate, palmoate and stearate.Also within the scope of the present invention, when they can be used,are the salts formed from bases such as sodium or potassium hydroxide.For other examples of pharmaceutically acceptable salts, reference canbe made to “Salt selection for basic drugs”, Int. J. Pharm. (1986),33:201-217.

The pharmaceutical composition can be in the form of a solid, forexample powders, granules, tablets, gelatin capsules, liposomes orsuppositories. Appropriate solid supports can be, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine and wax. Other appropriate pharmaceuticallyacceptable excipients and/or carriers will be known to those skilled inthe art.

The pharmaceutical compositions according to the invention can also bepresented in liquid form, for example, solutions, emulsions, suspensionsor syrups. Appropriate liquid supports can be, for example, water,organic solvents such as glycerol or glycols, as well as their mixtures,in varying proportions, in water.

The invention also provides the use of a compound of formula (I) or(I′), or a pharmaceutically acceptable salt thereof, for the preparationof a medicament intended to treat inflammatory disorder.

The invention includes compounds, compositions and uses thereof asdefined, wherein the compound is in hydrated or solvated form.

In comparison to the prior art the improvement of the present inventionlies in the unexpected observation that3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one has superiorADME properties compared to the general classes of lactam BSCIs whichhave been described previously (such as, for example, the Internationalapplications supra). Although such compounds were reported as havingacceptable pharmacodynamic properties (that is, they have a potentanti-inflammatory effect in vivo as a result of their BSCI activity),and it was inferred that they must have acceptable pharmacokinetic, andhence ADME, properties, nevertheless direct assessment of the ADMEproperties suggest that3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one is notably andunexpectedly superior (see the examples below).

In particular, while previous in vitro stability studies in serumsuggested that lactam BSCIs were considerably better than the earlierimide BSCIs (see, for example, WO99/12968), as reported in theliterature (for example Fox et al. J. Med. Chem. 2005 48:867-74), it isnow clear that many (or indeed most) of the lactam class of BSCIs aresubject to unwanted metabolism in vivo. We have made and tested morethan a dozen BSCIs of the acylaminolactam class, and with the exceptionof 3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one, all of thelactam class BSCIs tested to date are subject to rapid liver metabolism(either cytochrome P450 mediated hydroxylation and/or phase IImetabolism).

At least partly as a consequence of the reduced in vivo metabolism, theoverall clearance of3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one is markedlylower than for the other lactam BSCIs tested. As a result, the exposurefollowing a single oral dose is more than 10-fold higher for3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one.3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one is thereforemore suitable for use as a human pharmaceutical, particularly wherechronic oral exposure is required for efficacy, than most (if not all)of the lactam BSCIs previously disclosed.

Prior art peptides (such as NR58-3.14.3) have the disadvantages that:(a) they are expensive and require solid phase synthesis (at least forthe longer ones) and (b) they clear very quickly via the kidneys and (c)they are generally very much less potent (>25 fold less potent in vitroand >10,000 fold less potent in vivo).

The prior art aminoglutarimides are cheap, not cleared quickly via thekidneys and more potent in vitro but they are extremely unstable inserum (as a result of an enzymatic opening of the imide ring; see, forexample, Fox et al. J. Med. Chem. 2005 48:867-74). As a resultaminoglutarimide BSCIs, such as (S)-3-(undec-10-enoylamino)glutarimide,are at least 250-fold less potent in vivo than2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one, even in models ofacute inflammation (such as LPS-induced endotoxemia marked by systemicTNF-α production, where the impact of compound stability and ADMEproperties is least apparent).

Another series of structurally related (but functionally largelydissimilar) compounds which have been described in the literature arebacterial autoinducer compounds typically based around the 6-memberedhomoserinelactone structure, usually with a 3-oxo-acyl side chain (forexample, see Bycroft et al. U.S. Pat. No. 5,969,158 which claims a broadrange of such compounds). Interestingly, although such disclosuresinclude generic formulae that encompass lactams as well as lactones, fewif any of the exemplified compounds with bacterial autoinducerproperties have lactam headgroups. All such compounds (but particularlythose with a lactone headgroup and/or a 3-oxoacyl tail group) are knownto be relatively unstable limiting their applications as medicaments.

In contrast, 3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one,is cheap to synthesise (and the method disclosed here allowsstraightforward synthesis even on a Kg scale), and shows excellentmetabolic stability not only in isolated serum in vitro (a propertyshared with the whole lactam class of BSCIs disclosed previously) butalso in vivo. As a result, the compounds of the present invention is(among those compounds studied extensively to date) uniquely optimisedfor the treatment of inflammatory diseases in man in terms of efficacy,potency and pharmaceutical properties such as ADME, pharmacokinetics,toxicology and safety pharmacology.

According to this invention, inflammatory disorders intended to beprevented or treated by the compounds of formula (I) or (I′) or thepharmaceutically acceptable salts thereof or pharmaceutical compositionsor medicaments containing them as active ingredients include notably:

-   -   autoimmune diseases, for example such as multiple sclerosis,        rheumatoid arthritis, lupus, irritable bowel syndrome, Crohn's        disease;    -   vascular disorders including stroke, coronary artery diseases,        myocardial infarction, unstable angina pectoris, atherosclerosis        or vasculitis, e.g., Behçet's syndrome, giant cell arteritis,        polymyalgia rheumatica, Wegener's granulomatosis, Churg-Strauss        syndrome vasculitis, Henoch-Schönlein purpura and Kawasaki        disease;    -   viral infection or replication, e.g. infections due to or        replication of viruses including pox virus, herpes virus (e.g.,        Herpesvirus samiri), cytomegalovirus (CMV), hepatitis viruses or        lentiviruses (including HIV);    -   asthma, and related respiratory disorders such as allergic        rhinitis and COPD;    -   osteoporosis; (low bone mineral density);    -   tumor growth;    -   organ transplant rejection and/or delayed graft or organ        function, e.g. in renal transplant patients;    -   a disorder characterised by an elevated TNF-α level;    -   psoriasis;    -   skin wounds and other fibrotic disorders including hypertrophic        scarring (keloid formation), adhesion formations following        general or gynaecological surgery, lung fibrosis, liver fibrosis        (including alcoholic liver disease) or kidney fibrosis, whether        idiopathic or as a consequence of an underlying disease such as        diabetes (diabetic nephropathy);    -   disorders caused by intracellular parasites such as malaria or        tuberculosis;    -   neuropathic pain (such as post-operative phantom limb pain,        post-herpatic neuralgia etc)    -   allergies; or    -   Alzheimer's disease.

According to this invention, further inflammatory disorders include:

-   -   ALS;    -   antigen induced recall response    -   immune response suppression.

These clinical indications fall under the general definition ofinflammatory disorders or disorders characterized by elevated TNFαlevels.

It should be noted, for the avoidance of doubt, that the primarymechanism of action of the BSCIs, including the compounds claimedherein, is on the immune system. Consequently, the claimed beneficialeffects on diseases such as viral infection and/or replication andtumour growth (conditions which, of themselves, are not primarilydiseases of the immune system) results from the consequential effects ofmodulating the immune system on the infection and/or replicationpatterns of the virus, or on the growth and spread of the tumour. SinceBSCIs, including the compounds claimed herein, do not (in general)directly affect viral replication or tumour growth, they would beexpected to have no effect at all in an isolated system (for example, inan in vitro infection of a cultured cell line, or in a tumour cell lineproliferation assay) where an intact and functioning immune system isabsent. Consequently, prior information relating to the effects of anycompounds in such isolated systems cannot inform the development ofBSCIs, which act on the immune system.

The invention also provides a method of treatment, amelioration orprophylaxis of the symptoms of an inflammatory disease (including anadverse inflammatory reaction to any agent) by the administration to apatient of an anti-inflammatory amount of a compound, composition ormedicament as claimed herein.

Administration of a medicament according to the invention can be carriedout by topical, oral, parenteral route, by intramuscular injection, etc.

The administration dose envisaged for a medicament according to theinvention is comprised between 0.1 mg and 10 g depending on theformulation and route of administration used.

According to the invention, the compounds of general formula (I) or (I′)can be prepared using the processes described hereafter.

Preparation of the Compounds of General Formula (I) or (I′)

All the compounds of general formula (I) or (I′) can be prepared easilyaccording to general methods known to the person skilled in the art.

Compound (I) is a colourless crystalline compound that can be made fromornithine and 2,2-dimethylpropionoyl chloride. For the synthesis of (I′)enantiomerically pure (S)-ornithine would be used. A ring closurestarting with either ornithine or its methyl ester is possible. Theamino acid can be esterified in dry methanol by in situ generation ofHCl using trimethylsilyl chloride. Alternatively, isolated ester can bering closed, in either case using triethylamine. The crude product canthen be acylated after a solvent exchange.

The acylaminolactam product (I) has significant water solubility and asa result, the acylation conditions used for related, more hydrophobic,products (see, for example, WO2006/134385) was unsatisfactory. The useof three equivalents of sodium carbonate as base resulted in theformation of significant precipitation of sodium hydrogen carbonateby-product unless a large amount of water was used (>4 mL/mmolornithine). At these concentrations the extraction of the product intodichloromethane is not efficient. Therefore the three equivalents ofsodium carbonate were replaced with 2.5 equivalents of potassiumhydroxide (which neutralises the 2.5 equivalents of triethylaminehydrochloride generated in the ring closure step). With this basesignificantly less water can be used (less than 1 mL/mmol ornithine)(final pH 8-9). Extraction of the aqueous layer with EtOAc (3×2 mL/mmolornithine) and recrystallisation from EtOAc (0.5 mL/mmol, hot) and 40-60petroleum (5 mL/mmol) produced a first crop in 43% yield (4.25 g from 50mmol ornithine).

Note that if the pH of the aqueous layer is too low during the work-upthen small quantities of triethylamine hydrochloride will be extractedinto the EtOAc layer. Attempts at washing this EtOAc solution with waterwill extract significant quantities of the lactam product (I) along withthe amine hydrochloride. This can be avoided by raising the pH of theaqueous layer to 12 (for example by addition of roughly one equivalentof KOH with respect to acid chloride) before the EtOAc extraction isattempted, then only triethylamine free base is extracted along with thelactam product (I), which can be removed more easily by evaporation orduring recrystallisation.

The following preferred synthetic route is therefore provided:

(S)-Ornithine monohydrochloride (50 mmol) is suspended in dry methanol(100 ml) and trimethylsilyl chloride (75 mmol) added. The reaction isheated at reflux for 24 hours. Triethylamine (150 mmol) is then addedand the reaction is heated at reflux for 48 hours. The methanol is thenremoved under reduced pressure (optionally, toluene may be added in thelatter stages to facilitate removal), and the residue is dissolved inwater (20 mL), with KOH (125 mmol) added.

The mixture is cooled to 0° C. then 2,2-Dimethylpropionyl chloride (50mmol) is added slowly and the reaction stirred for 18 hours, whilewarming to ambient temperature. Solid KOH (50 mmol) is then added andonce it has dissolved the reaction is extracted with EtOAc (3×100 mL).The combined organic layers are quickly dried over a combination ofK₂CO₃ and Na₂SO₄, and reduced under low pressure. The solid residue isthen recrystallised from EtOAc (25 mL)/40-60 petroleum ether (200-250mL) to give the lactam (I′) as a crystalline solid (greater than 50%yield).

The identity and purity (>95%) of the product was then confirmed byproton NMR spectroscopy OH (400 MHz, CDCl₃) 6.63 (1H, br s, NH), 6.01(1H, br s, NH), 4.20 (1H, dt, J 11, 5.5, CHNH), 3.40-3.31 (2H, m,CH₂NH), 2.61 (1H, dq, J 13, 4.5, CH₂), 1.97-1.88 (2H, m, CH₂), 1.50 (1H,dddd, J 13, 12, 9.5, 7.5, CH₂), 1.22 (9H, CH₃).

DEFINITIONS

The term “about” refers to an interval around the considered value. Asused in this patent application, “about X” means an interval from Xminus 10% of X to X plus 10% of X, and preferably an interval from Xminus 5% of X to X plus 5% of X.

The use of a numerical range in this description is intendedunambiguously to include within the scope of the invention allindividual integers within the range and all the combinations of upperand lower limit numbers within the broadest scope of the given range.Hence, for example, the range of 0.1 mg to 10 g specified in respect of(inter alia) the dose of3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one to be used isintended to include all doses between 0.1 mg and 10 g and all sub-rangesof each combination of upper and lower numbers, whether exemplifiedexplicitly or not.

As used herein, the term “comprising” includes both comprising andconsisting. Consequently, where the invention relates to a“pharmaceutical composition comprising as active ingredient” a compound,this terminology is intended to cover both compositions in which otheractive ingredients may be present and also compositions which consistonly of one active ingredient as defined.

Unless otherwise defined, all the technical and scientific terms usedhere have the same meaning as that usually understood by an ordinaryspecialist in the field to which this invention belongs. Similarly, allthe publications, patent applications, all the patents and all otherreferences mentioned here are incorporated by way of reference.

The following examples are presented in order to illustrate the aboveprocedures and should in no way be considered to limit the scope of theinvention.

FIGURES

FIG. 1 (panels A to E) shows the time-concentration graphs for the fivecompounds (I′) to (V) tested, following administration of a single 3mg/kg dose in 1% CMC via the oral route to rats. The three lines foreach compound represent three replicate animals. The Y-axis representsconcentration in units of ng/ml (0-3000); the X-axis represents time inunits of minutes (0-480).

FIG. 2 shows the major metabolites identified by LC-MS-MS (in full scanmode) on pooled urine collected over 24 hrs from rats exposed to asingle oral dose of each of the five compounds (I′) to (V) tested,administered in 1% CMC at 3 mg/kg. Note that a major metabolite ofcompound (IV) has been identified but its structure was not elucidatedby comparison of fragmentation/re-arrangement patterns in the publiclyavailable Metabolite ID databases. Although the concentrations of theindividual metabolites were not determined, they are shown inqualitative order of abundance in urine, with the most abundant specieson the left of each row.

FIG. 3 (panels A-F) shows the current versus time graphs for cellsexpressing the hERG gene product, when exposed to either vehicle or tothe five compounds (I′) to (V), each in a separate experiment. In eachexperiment, replicate cells were exposed to the positive controlcompound, which completely blocks hERG-transduced current. The Y-axisrepresents current in units of nA; the X-axis represents time in unitsof seconds. Panel G shows the hERG tailcurrent (the area under the timeversus current graphs in panels A-F) for replicate cells exposed to eachcompound or to 0.1% DMSO vehicle alone (Veh) or to the positive controlcompound E-4031 (+ve). The Y-axis of the histogram in panel G representspercentage inhibition of the hERG tail current relative to untreatedcells.

FIG. 4 shows a typical binding curve for compound (I′). In thisexperiment, each reaction received 10 nM labelled BN83250 (a lactam BSCIwhich binds to the same receptor as compound (I′)), together withvarious competitors (100 μM-1 pM). The total specific binding was theamount displaced by a large excess (100 μM). Each bar represents themean of three replicate determinations, with whiskers representing SEM.The Y-axis represents the radioactive counts bound in each experimentsin units of counts per minute (cpm). The upper dashed line representsthe total binding under the conditions of the experiment, while thelower dashed line represents the non-specific binding; the specificbinding is represented by the distance between the dashed lines.

FIG. 5 shows the profile of cross-reactivity for each of the fivecompounds (I′) to (V) (panel A: (II); B: (III); C: (V); D: (I′) and E:(IV)) against a panel of 75 different receptors that compose the CEREPpanel (see text). The compounds were tested at a single concentration(10 μM), and the inhibition of binding (Y-axis of each histogram) for aknown ligand to each of the 75 receptors is reported (−100% thereforerepresents a 2-fold increase in binding of the specific ligand in thepresence of the test compound). All reactions were performed induplicate, and the bars represent the mean (no estimate of the replicateerror is shown to simplify the graphs). Only a 50% or greater inhibition(or stimulation) of binding (representing an ED50 below 10 μM for theinteraction of the test compound with the particular receptor) wasconsidered statistically and biologically significant in this screeningassay. For the five compounds tested here, only one interaction (that ofcompound (II) with the NK2 receptor, marked by the arrowhead in panel A)was considered potentially significant, although even this interactionwas weak (estimated ED50 5-10 μM).

FIG. 6 (panels A-E) shows representative dose response curves for theinhibition of chemokine-induced leukocyte migration in vitro, for eachof the five compounds (I′) to (V) in the ChemoTx™ transwell migrationassay. In each experiment, THP-1 cells were induced to migrate using amaximally effective dose of the chemokine MCP-1 in the presence orabsence of various doses (from 10 μM to 1 μM) of each compound. Anappropriate vehicle control was used in each experiment. The percentageinhibition of MCP-1 induced migration (calculated as the number of cellsmigrated in the presence of MCP-1 minus the number of cells migratedwith MCP-1 omitted from the lower chamber) at each concentration of eachtest compound is shown as the mean of triplicate determinations, withwhiskers representing SEM. The ED50 was estimated by linearinterpolation of the presented graphs. The Y-axis of each graphrepresents the percentage inhibition of MCP-1 induced migration; theX-axis represents the concentration of test compound present in units ofnM (0.01-1000).

FIG. 7 shows representative dose response curves for the inhibition ofLPS induced TNF-α production in vivo in a murine model of sub-lethalendotoxemia. In each experiment, groups of six mice receivedpre-treatment with the five compounds (panel A: (II); B: (III); C: (V);D: (I′) and E: (IV)) at various doses, via either the oral route(circles) or subcutaneous route (triangles). 30-60 mins later, animalswere challenged with LPS via the intraperitoneal route, and serum wasprepared from a terminal bleed 3 hours later. The level of TNF-α in theblood was measured by ELISA, and the degree of inhibition of LPS-inducedTNF-α production (calculated as the concentration of TNF-α in miceexposed to LPS minus the concentration of TNF-α in mice which received asham exposure to endotoxin-free PBS) is shown on the Y-axis of eachgraph as the mean of six animals, with whiskers representing SEMs. Theconcentration of TNF-α in mice receiving LPS but no BSCI treatment wastypically 5,000 to 6,000 pg/ml on average (compared to <10 pg/ml inunchallenged mice). The ED50 was estimated by linear interpolation ofthe presented graphs. The X-axis of each graph represents the dose ofeach compound administered to each mouse in the group in units of mg(1E-07 to 1).

FIG. 8 shows the effect of compound (I′) on lung inflammation in arepresentative experiment, as assessed by cell counts in BAL fluid in arodent model of asthma. Bars represent mean cell counts, shown on the Yaxis in units of 10⁶ cells, for groups of 5 animals with whiskersrepresenting SEM; *p<0.01 versus ‘sensitised’ using Student's unpairedt-test assuming equal variance, with two tails). The horizontal linerepresents the average number of leukocytes present in BAL fluid fromthe lungs of unchallenged rats. All rats in the remaining three groupsreceived the same sensitisation and challenge regimen, but were eithertreated with vehicle only (‘Sensitised’), or with compound (I′) at 0.3mg/kg bodyweight or with monteleukast (‘Singulair™’) at 30 mg/kgbodyweight, all administered daily by oral gavage.

FIG. 9 shows the effect of compound (I′) on T-helper cell polarisationin a representative experiment, as assessed by flow cytometricdetermination of IFN-γ (a Th1 marker cytokine) and IL-4 (a Th2 markercytokine) production by CD4+ splenocytes in a rodent model of asthma.Bars represent mean Th1/Th2 ratios, shown on the Y-axis, for groups of10 animals, whiskers represent SEM; *p<0.05 versus unchallenged rats;†p<0.05 versus sensitised and challenged rats, in both cases usingStudent's unpaired t-test assuming equal variance, with two tails). Allrats (except the ‘BN rats’ group, which was not exposed to ovalbumin)received the same sensitisation and challenge regimen, but were eithertreated with vehicle only (‘Sensitised+vehicle’), or with compound (I′)at 0.3 mg/kg bodyweight or with monteleukast (‘Singulair™’) at 30 mg/kgbodyweight, all administered daily by oral gavage.

EXAMPLES

In each of the following examples3-(2′,2′-dimethylpropanoylamino)-tetrahydropyridin-2-one (compound I′)has been compared with a range of other lactam BSCIs which were selectedto be representative of the various subclasses. For example,3-(adamantane-1-carbonylamino)-caprolactam (II) was selected as typicalof the subclass of polycycloacyl lactam BSCIs (such as those disclosedpreviously in WO2006/016152).

Similarly, 3-(1′-methylcyclohexylcarbonylamino)-caprolactam (III) wasselected as typical of the subclass of monocycloacyl lactam BSCIs (suchas those disclosed previously in WO2006/134384).

The compound 3-(1′,1′-dimethylethylsulfonylamino)-caprolactam (IV) wasselected as typical of the subclass of BSCIs with simple (noncyclic)alkyl side chains (such as those disclosed previously in WO2005/053702),as well as those with a sulfonylamino linker (as opposed to the carbonamide linker in the remaining compounds selected).

The final compound selected,3-(3′-hydroxyadamantyl-1-carbonylamino)-caprolactam (V) was typical ofBSCIs with a substituted acyl side chain (whether simple linear,branched, mono- or polycyclo in structure).

It is important to note that all of these BSCI compounds (II) through(V) have been specifically disclosed previously, and all have potentBSCI activity in vitro (ED50<1 nM for the inhibition of MCP-1 inducedTHP-1 cell migration). All have excellent stability in serum, in vitro,and from a theoretical perspective are all excellent candidates fordevelopment as human pharmaceuticals with anti-inflammatory propertiesin vivo.

All of the compounds studied here (I′, II, III, IV and V) were tested inthe (S) configuration at the lactam stereocentre.

Example 1 Pharmacokinetics Following a Single Dose

Compounds were administered as a single dose (either 1 mg/kg bodyweightin 5% DMSO via the intravenous route or 3 mg/kg in 1%carboxymethylcellulose via the oral route) to three adult rats (usingdifferent rats for each compound and each route of administration).

Blood was then sampled at various time points (including just prior todose administration) out to 24 hrs post dose, and the level of thevarious compounds was assessed using a validated LC-MS/MS assay.Briefly, 3-5 μl of the deproteinised sample was applied to a WatersAtlantis (C18 20×2.1 mm, 3 μm bead size) reverse phase chromatographycolumn equilibrated in 0.1% formic acid in 95:5 water:actetonitrile.Over 3.5 minutes bound material was gradient eluted reaching 0.1% formicacid in 5:95 water:acetonitrile, followed by a step gradient back to0.1% formic acid in 95:5 water:acetonitrile. The column eluent was thendirected to an Applied Biosystems API 4000/3200 QTrap MS/MS massspectrometer, with a Turbolonspray™ ion source operating in positive ionmode. The interface temperature was set at 650° C., with a 40 ms dwelltime for each MRM transition, monitoring the following ions:

Analyte Q1 Mass (amu) Q3 Mass (amu) (II) 291.14 135.1 (III) 253.16 97.0(V) 307.15 151.1 (I′) 199.15 57.2 (IV) 249.15 129.3 Internal standard213.19 57.1

The internal standard in each determination was the related compound(S)-3-(2′,2′-dimethylpropanoylamino)-caprolactam, which was spiked intothe sample prior to deproteinization. The lower limit of quantitation(LLOQ) for this assay was 2.4 ng/ml for each compound except (I′), whereit was 38.1 ng/ml.

Following LC-MS/MS analysis of each collected sample, thepharmacokinetic disposition of the compound was modelled using Kineticasoftware, a well known software package for such applications.

Results

The individual concentration versus time graphs for each rat treatedwith each compound via the oral route are shown in FIG. 1. It isimmediately obvious that of these five structurally diverse lactam BSCIsonly (IV), (V) and (I′) achieve any appreciable oral exposure, and thatof these (I′) is substantially better than the others.

The parameters of a simple one-compartment pharmacokinetic model areshown in Table 1. Firstly, this demonstrates the superior exposureachieved with (I′)—nearly 20-fold higher than the next best compound,(V). The reason for this superior exposure (which is calculated as thearea under the concentration versus time curve, in the units ofmin·ng/ml) is also evident: the clearance of (I′), which is defined asthe theoretical volume of blood which is completely cleared of drug eachminute in the units ml/min/kg, is more than 10-times lower than for theother lactam BSCIs.

TABLE 1 Pharmacokinetic parameters for structurally diverse lactamBSCIs. Clearance Exposure F (%) t½ (mins) (ml/min/kg) Vss (L/kg) (min ·ng/ml) (I′) 69 196  2.6 0.7 939,000 (II) <1 16 84 0.8 477 (III) 5 11 550.6 2,900 (IV) 45 19 32 0.6 41,400 (V) 59  24* 31 1.4 57,100 Oralbioavailability (F, %), dominant plasma half-life (t½, mins), clearance(ml/min/kg), volume of distribution (Vss, L/kg) and exposure (AUC0->infinity, min · ng/ml) from a simple one-compartment pharmacokineticmodel for each compound, averaged across three rats. *24 mins is thedominant half-life for (V) accounting for the clearance of more than 95%of the injected dose; the minor t½β was 110 mins. In all cases the Cmaxwas achieved within 30 mins, consistent with optimal absorption.

The clearance of (II) and (III) approximates to the liver blood flow ofthe rat, strongly suggesting that both these compounds are metabolisedalmost completely on first pass through the liver. Similarly theclearances of both (IV) and (V) exceed renal blood flow in the rat bysome 3-4 fold, again suggesting substantial metabolic clearancepresumably also liver mediated. In marked contrast, the clearance of(I′) at 2.6 ml/min/kg is less than half of renal blood flow (typicallyquoted as 7-9 ml/min/kg), which suggests minimal metabolic clearance.Since (I′) is extremely water soluble, with a volume of distributionconsistent with free equilibration in total body water (0.7 L/kg), it islikely that the clearance below renal blood flow represents reabsorptionwith water in the renal distal tubule (rather than, for example, reducedexposure to the kidney due to sequestration into lipophiliccompartments).

Consistent with the much lower clearance for (I′) compared with theother compounds, (I′) has a substantially longer plasma half-life (morethan 3 hours, compared with less than half-an-hour for the other fourcompounds).

Not all of the BSCIs are observed to have oral bioavailability, eventhough all five of the chosen compounds were known to have oralbioactivity on acute inflammation end-points. This likely reflects therapid liver-mediated metabolism of (II) and (III), which are absorbedefficiently but converted on first pass through the liver to metaboliteswhich retain some activity as BSCIs (see example 2).

Based on this pharmacokinetic analysis, it will be obvious to thoseskilled in the art that despite the similar chemical stability and invitro stability in isolated serum for these five compounds, as well astheir similar predicted properties on theoretical grounds, nevertheless(I′) is markedly superior to all the others. In particular, theclearance of the compound is much lower, probably reflecting a reducedpropensity to liver-mediated metabolism, resulting in a 10-fold longerplasma half-life and almost 20-fold better oral exposure than the nextbest compound examined.

In a separate experiment, (I′) was compared with the second bestcompound (V) for pharmacokinetic parameters in a different (non-rodent)species, the dog. Single doses (1 mg/kg in 5% DMSO intravenously or 3mg/kg in 1% CMC per os) were administered to a single dog for eachcompound in a simple crossover design with 1 week washout between thetwo routes of administration. The results are shown in Table 2.

TABLE 2 Comparison of the pharmacokinetic parameters for the best twolactam BSCIs in rat and dog. Intravenous PK (1 mg/kg) Half-lifeClearance Vss Compound Species (mins) (ml/min/kg) (L/kg) (V) Rat 24 330.8 Dog 66 7.4 0.6 (I′) Rat 196 2.6 0.7 Dog 236 1.9 0.6 Oral PK (3mg/kg) AUC Tmax Half-life Bioavailability (min · ng/ Compound Species(mins) (mins) (%) ml) (V) Rat* 15 24 59 57100 Dog 60 67 59 240000 (I′)Rat 15-120 226 81 939000 Dog 60 217 75 1210638 The pharmacokinetics indog are broadly similar to rat. In both species, compound (I′) havingconsiderably lower clearance, longer plasma half-life and hence greaterexposure (AUC in min · ng/ml). In each case the predominant half-life(the faster t½α) is responsible for clearing more than 95% of theinjected dose.

These observations indicate that the superior pharmacokinetic propertiesof (I′) are not species specific and are consequently very likely alsoto be observed in humans.

Example 2 Identification of the Primary Metabolites

Urine was collected from rats exposed to a single oral dose (3 mg/kg in1% CMC) of each compound over a 24 hour period in metabolic cages. Thepooled urine sample was then subjected to full scan mass spectralanalysis, using the same LC-MS conditions as described in example 1above. Further MS-MS analysis of the product ions was then performed,and likely fragmentations/re-arrangements assigned from the publiclyavailable Metabolite ID database.

For the major metabolites, the assigned structures were confirmed bysynthesis of authentic samples, using methods well known in the art,which were subject to LC-MS-MS analysis under the same conditions as theurine samples.

Note that metabolite ID studies provide only qualitative estimates ofthe relative amounts of the different metabolites present, andseparately validated assays with appropriate internal standards would berequired to quantitate each metabolite species.

Results

The detected metabolites, in rank order of concentration detected areshown in FIG. 2 for the five compounds analysed. It is important to notethat the methodology used here is not necessarily exhaustive, andfurther (particularly minor) metabolites may also be present which werebelow the detection limits for the methods applied here. As a generalrule, it can be assumed that metabolites representing 10% or more of theinjected dose will be detected (though not necessarily structurallyidentified) by the methods used here).

For compounds (II) and (III) the major route of metabolism is cytochromeP450-mediated hydroxylation, consistent with the rapid clearance at arate approaching liver blood flow (see example 1 above). The major siteof hydroxylation in both compounds is on the cycloalkyl tail group, witha second (slower) hydroxylation occurring on the lactam head group. Notethat the lactam hydroxylation products appeared in the MS-MS at −2 amu(as opposed to +16 amu) because of the instability of the 7-hydroxyadducts in the electrospray source.

For compound (II) the dihydroxylated product was present in sufficientquantity to be detected in urine, whereas for compound (III) nodihydroxylation product was detected. In both cases, it is likely thatadditional minor products were also formed below the level of detectionof the method applied (for example, the 3,5 dihydroxy and 3,5,7trihydroxy adamantyl derivatives of (II), as well as the glucuronidatedadducts of both hydroxy-(II) and hydroxy-(III), particularly since theglucuronidated adduct of (V) was detected).

For compound (V) the glucuronidated compound was the major metabolite,although in rat only a minor fraction of the glucuronate is eliminatedin urine, the bulk passing into faeces (in marked contrast to humans,where this glucuronidated adduct would be primarily excreted in urine).It is possible that other phase II metabolites (such as the3′-O-sulphate) were also formed, but only at levels (at least in urine)too low to be detected by the methods used here. As for compounds (II)and (III), a small amount of product hydroxylated on the lactam headgroup was also detected (again primarily as a −2 amu product ion):

For compound (IV) the major metabolite could not be identified, althoughthe loss of parent compound (see Table 3) was clearly consistent withthe formation of an unidentified metabolite (less than 10% of theinjected dose of (IV) was recovered unchanged. Given that compound (IV)was the only agent containing a sulfonamide linkage it is plausible, butcurrently unproven, that metabolic cleavage (or other modification) ofthe linker was occurring. Once again, a small amount of hydroxylation atthe lactam head group was also observed.

In marked contrast to all the other compounds, no significantmetabolites of (I′) were detected in urine, consistent with theappearance of the majority of the injected dose in the urine inunchanged form (see Table 3) and the clearance rates at or below renalblood flow (see example 1 above). This lack of formation of metabolitesis a major, and unexpected, advantage of (I′) over the other compoundstested here for development as a human pharmaceutical and is, at leastin part, responsible for the superior pharmacokinetic propertiesdescribed in example 1 above.

To provide a quantitative estimate of the degree of metabolism sufferedby each of the compounds tested, the amount of unchanged parent compoundin urine was measured using the same validated LC-MS assay described inexample 1, using (S)-3-(2′,2′-dimethylpropanoylamino)-caprolactam as aninternal standard. The results are shown in Table 3. In addition, thelevel of compounds in various target tissues was also determined.

TABLE 3 Distribution of compounds into various tissues 24 hrs after asingle dose in rat. Urine Compound Heart Lung Kidney Liver Muscle Brain(ng/ml) (II) nd Nd nd nd nd nd nd (III) nd Nd nd nd nd nd nd (V) nd Ndnd nd nd nd 16300 (I′) 5.3 21.9 5.1 3.6 3.1 1.6 24567 (IV) nd Nd nd ndnd nd  1010 Only compounds (I′), (V) and (IV) were detectable in urineat 24 hrs after a single oral dose (3 mg/kg in 1% CMC). Of these,compound (I′) underwent significantly less metabolism (more than 60% ofthe injected dose was recovered in urine). Furthermore, only compound(I′) could be detected in any of the other tissues examined 24 hrs aftera single dose. This likely reflects both the superior distribution andincreased exposure associated with (I′) compared to the other compoundstested here. nd = not detected; LLOQ = 2.4 ng/g

The much lower rate of metabolism of (I′) compared to the othercompounds demonstrates that, unexpectedly, (I′) is markedly superior tothe wide range of lactam BSCIs previously disclosed for development as ahuman pharmaceutical. This reduced metabolism (and hence improved ADMEproperties) likely accounts for the dramatically superiorpharmacokinetic properties shown in example 1 above. Furthermore, sinceBSCIs are intended for development as anti-inflammatory agents targetinginappropriate leukocyte recruitment into a wide range of tissues, theunexpected finding that (I′) is found in all tissues of the body tested24 hrs after a single dose, whereas all the other lactam BSCIs testedwere not, unequivocally demonstrates the particular utility of thisnovel compound.

Example 3 Safety Pharmacology

The five compounds were subjected to a standard AMES test to assesslikely genotoxicity. Three His-auxotroph strains of S. Typhinurium (TA102, TA98 and TA 100) were treated with each of the compounds at 5concentrations (up to 5 mg/ml) in the presence and absence of S9 ratliver microsomal metabolising system. The number of revertant colonieswas then determined by plating on trace-His minimal media.

The results (Table 4) show that none of the five compounds significantlyincrease revertant colony formation (with or without metabolicactivation) in any of the strains tested.

TABLE 4 Revertant colony formation in AMES test. Com- TA100 + TA102 +TA98 + pound TA100 S9 TA102 S9 TA98 S9 (II) 0.59* 0.73 0.50* 0.93 0.58*0.60* (III) 0.79 0.81 0.69 0.88 0.68 0.55 (V) 0.78 0.88 0.93 1.09 0.960.91 (I′) 0.97 0.95 0.82 0.93 2.00** 1.23 (IV) 0.89 0.90 0.85 0.88 0.820.90 +ve 6.89 7.18 5.97 2.65 9.68 19.05 control None of the compoundstested caused a significant increase in revertant colony formation atany of the concentrations tested (data for the top dose only is shown).Note that compound (II) caused inhibition of bacterial lawn growth at 5mg/ml. At 5 mg/ml: *= sparse bacterial background lawn **= low controlvalue in this experiment = significant increase in revertant colonies

In a separate experiment, all five compounds were tested for interactionwith the hERG ion channel. Compounds which interact with hERG are atrisk of causing QT prolongation and potentially fatal cardiacarrhythmia. Compounds which inhibit hERG tail current by more than 50%at 10 μM are generally considered high risk for development as humanpharmaceuticals.

HEK239 cells stably transfected to express hERG were perfused with bathsolution containing the compounds at 10 μM (0.1% DMSO). hERG tailcurrents from three cells were then recorded by patch-clamp analysisfollowing depolarisation to +20 mV for 5 s. The potency of the hERGinteraction was then determined in a 4-point dose-response curve for anycompounds showing significant modulation at 10 μM.

The results (FIG. 3) show that none of the five compounds significantlyinteracted with the hERG channel at 10 μM.

We conclude that, from a safety pharmacology perspective, all five ofthe compounds, including (I′) are equally suitable for development ashuman pharmaceuticals. In particular, the considerably superior ADME andpharmacokinetic properties of (I′), illustrated in Examples 1 and 2above, are not accompanied by correspondingly worse safety pharmacologyprofiles.

Example 4 General Pharmacology

The general pharmacology of the five compounds was assessed, bothagainst the specific target receptor, and against a wide variety ofother receptors, many related in structure to the target receptor.Specific binding to the target receptor was assessed by competition forthe binding of [³H]-BN83250 (BN83250 is(S)-3-(2′,2′-dimethyldodecanoylamino)-caprolactam; Fox et al. J MedChem. 200; 48(3):867-74; an agent known to bind to the same targetreceptor as the lactam BSCIs disclosed here). Binding to non-targetreceptors was assessed by competition for the binding of variousspecific radioligands for other receptors which are well known in theart.

For specific binding the human myelomonocytic cell line was resuspendedin binding buffer (20 mM HEPES, 150 mM NaCl, pH 7.4; 10⁶ cells perreaction) at 4° C. in the presence of 10 nM [³]-BN83250 (from 1 μM stockin 100% ethanol; 30 Ci/mmol) and various competitors (1% DMSO maximumvehicle concentration). Reactions were incubated for 2 hours on ice,then filtered through GF/C filters pre-soaked in 0.5% polyethyeneimine.Unbound material was washed away with 5×5 ml ice cold washing buffer (20mM HEPES, 150 mM NaCl, pH 7.4) under a slow vacuum. These conditionshave previously been shown to achieve equilibrium binding, with at least80% of the binding specific (competable with 10 μM cold BN83250).

Competition for the specific [³H]-BN83250 with compounds (I′), (II) and(V) was then determined at various concentrations from 1 μM to 10 μM.Compounds (III) and (IV) were not examined in these experiments. Atypical competition binding curve for (I′) is shown in FIG. 4.

Non-linear modelling was then applied to the competition binding curvesfor the various compounds, in order to compare their properties asagents binding to the target receptor (defined as the site of specificinteraction of BN83250). The parameters of the resulting models aregiven in Table 5.

TABLE 5 Non-linear modeling of competitive binding curves. Functional Kaat target ED50 versus receptor Compound MCP-1 (pM) (pM) Hill slope (II)80 8,200 −0.5 (III) 80 Not tested Not tested (V) 120 10,000 −0.5 (I′) 5050 −1.0 (IV) 800 Not tested Not tested It is important to note thatcompound (I′), in marked contrast to lactam BSCIs (II) and (V), showedideal and predictable binding to the target receptor. In particular, theapparent affinity for binding to the receptor was of a similar magnitudeto the functional ED50 value in migration inhibition assays. Similarly,the Hill Slope was approximately −1.0 (the theoretically expected valuefor a simple, non-cooperative competitive binding model), whereas theother lactam BSCIs showed considerably shallower Hill Slopes. The causeof the departure from ideal binding to the target receptor for compounds(II) and (V) is not known, but this difference further underlines theunexpected superiority of compound (I′).

Binding to non-target receptors was assessed using similar protocols,exploiting specific radioligands for each receptor which are well knownin the art. Each compound was tested for competition against thespecific binding of each ligand only at a single concentration (10 μM).Where the inhibition of binding was between 20% and 80%, the Ka for theinteraction was estimated. Where the inhibition was <20%, the compoundwas assumed to have no competitive interaction with the receptor. Wherethe inhibition was >80% the Ka was reported as <1 μM. Details of thereceptors screened, and the radioligands and cell types used in theassays, are available at www.cerep.fr

The results (FIG. 5) demonstrate that all of the lactam BSCI compoundstested are devoid of major cross-reactivities based on this panel of 75receptors (no interactions with an estimated Ka <1 μM were noted). Onlyone weak (but statistically significant) cross-reaction was noted(compound (II) with the NK2 receptor). On this basis, compound (I′) wasmarginally more specific for the target receptor than (II), but all thelactam BSCI compounds tested were suitable for development as humanpharmaceuticals based on their lack of off-target binding identified inthis high throughput screening assay format.

Example 5 Broad-Spectrum Chemokine Inhibition Activity In Vitro

The biological activity of the compounds of the current invention may bedemonstrated using any of a broad range of functional assays ofleukocyte migration in vitro, including but not limited to Boydenchamber and related transwell migration assays, under-agarose migrationassays and direct visualisation chambers such as the Dunn Chamber.

For example, to demonstrate the inhibition of leukocyte migration inresponse to chemokines (but not other chemoattractants) the 96-wellformat ChemoTx™ micro transwell assay system from Neuroprobe(Gaithersburg, Md., USA) has been used. In principle, this assayconsists of two chambers separated by a porous membrane. Thechemoattractant is placed in the lower compartment and the cells areplaced in the upper compartment. After incubation for a period at 37° C.the cells move towards the chemoattractant, and the number of cells inthe lower compartment is proportional to the chemoattractant activity(relative to a series of controls).

This assay can be used with a range of different leukocyte populations.For example, freshly prepared human peripheral blood leukocytes mayused. Alternatively, leukocyte subsets may be prepared, includingpolymorphonuclear cells or lymphocytes or monocytes using methods wellknown to those skilled in the art such as density gradientcentrifugation or magnetic bead separations. Alternatively, immortalcell lines which have been extensively validated as models of humanperipheral blood leukocytes may be used, including, but not limited toTHP-1 cells as a model of monocytes or Jurkat cells as model of naïve Tcells.

Although a range of conditions for the assay are acceptable todemonstrate the inhibition of chemokine-induced leukocyte migration (seefor example the advice provided by Frow et al. Med Res Rev. 2004;24(3):267-98 on the conditions required to interpret in vitro migrationassays), a specific example is hereby provided.

Materials

The transwell migration systems are manufactured by Neuroprobe,Gaithersburg, Md., USA. The plates used are ChemoTx™ plates (Neuroprobe101-8) and 30 μl clear plates (Neuroprobe MP30).

Geys' Balanced Salt Solution is purchased from Sigma (Sigma G-9779).Fatty acid-free BSA is purchased from Sigma (Sigma A-8806). MTT, i.e.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, ispurchased from Sigma (Sigma M-5655). RPMI-1640 without phenol red ispurchased from Sigma (Sigma R-8755).

The THP-1 cell line (European Cell Culture Collection) were used as theleukocyte cell population.

Test Protocol

The following procedure is used for testing the compound of the presentinvention for the ability to specifically block leukocyte migrationinduced by chemokines:

First, the cell suspension to be placed in the upper compartment isprepared. The THP-1 cells are pelleted by centrifugation (770×g; 4 mins)and washed with Geys Balanced Salt Solution with 1 mg/ml BSA (GBSS+BSA).This wash is then repeated, and the cells repelleted before beingresuspended in a small volume of GBSS+BSA for counting, for exampleusing a standard haemocytometer.

The volume of GBSS+BSA is then adjusted depending on the number of cellspresent so that the cells are at final density of 4.45×10⁶ cells per mlof GBSS+BSA. This ensures that there are 100,000 THP-1 cells in each 25μl of the solution that will be placed in the upper chamber of theplate.

To test a single compound for its ability to inhibit chemokine inducedmigration, it is necessary to prepare two lots of cells. The suspensionof THP-1 cells at 4.45×10⁶ cells/ml is divided into two pots. To one potthe inhibitor under test is added at an appropriate final concentration,in an appropriate vehicle (for example at 1 μM in not more than 1%DMSO). To the second pot an equal volume of GBSS+BSA plus vehicle asappropriate (e.g. not more than 1% DMSO) is added to act as a control.

Next, the chemoattractant solution to be placed in the lower compartmentis prepared. For example, MCP-1 is diluted in GBSS+BSA to give a finalconcentration of 25 ng/ml. This is divided into two pots, as for thecell suspension. To one pot, the test compound is added to the samefinal concentration as was added to the cell suspension, while to theother pot an equal volume of GBSS+BSA plus vehicle as appropriate (e,g.not more than 1% DMSO) is added. Alternatively, other chemokines may beused (SDF-1α at 7.5 ng/ml; RANTES at 50 ng/ml; IL-8 at 10 ng/ml usingneutrophils as the target cell population). In each case it is importantto determine (in a separate experiment) the concentration of eachchemokine which causes maximal stimulation of migration of the chosentarget leukocyte population. This maximal concentration must then beused in experiments to test the inhibitory activity of the compounds ofthe invention. Because chemokines typically induce leukocyte migrationwith a bell-shaped dose-response curve, the use of a sub- orsupra-maximal chemokine concentration can lead to artefactual results(for example, a compound which is a chemokine inhibitor can yield aparadoxical stimulation of leukocyte migration if a supra-maximalconcentration of chemoattractant is incorrectly selected for theexperimentation. Further illustrations of this important factor in thedesign of in vitro leukocyte migration experiments has are provided byFrow and colleagues (Med Res Rev. 2004; 24(3):276-98). In addition,non-chemokine chemoattractants may also be used to demonstrate thechemokine-selectivity of the biological activity of the compounds of theinvention (for example C5a at 25 ng/ml using neutrophils as the targetcell population or TGF-β1 at 10 ng/ml using THP-1 cells as the targetpopulation).

Note that the volume of liquid that needs to be added with the additionof the test compound needs to be taken into account, when establishingthe final concentration of MCP-1 in the solution for the lowercompartment and the final concentration of cells in the uppercompartment.

Once the chemoattractant solutions for the lower wells and cellsolutions for the upper chambers have been prepared, the migrationchamber should be assembled. Place 29 μl of the appropriatechemoattractant solution into the lower well of the chamber. Assaysshould be performed with at least triplicate determinations of eachcondition. Once all the lower chambers have been filled, apply theporous membrane to the chamber in accordance with the manufacturer'sinstructions. Finally, apply 25 μl of the appropriate cell solution toeach upper chamber. A plastic lid is placed over the entire apparatus toprevent evaporation.

The assembled chamber is incubated at 37° C., 5% CO₂, for 2 hours. Asuspension of cells in GBSS+BSA is also incubated under identicalconditions in a tube: these cells will be used to construct a standardcurve for determining the number of cells that have migrated to thelower chamber under each condition.

At the end of the incubation, the liquid cell suspension is gentlyremoved from the upper chamber, and 20 μl of ice-cold 20 mM EDTA in PBSis added to the upper chamber, and the apparatus is incubated at 4° C.for 15 mins. This procedure causes any cells adhering to the undersideof the membrane to fall into the lower chamber.

After this incubation the filter is carefully flushed with GBSS+BSA towash off the EDTA, and then the filter is removed.

The number of cells migrated into the lower chamber under each conditioncan then be determined by a number of methods, including directcounting, labelling with fluorescent or radioactive markers or throughthe use of a vital dye. Typically, we utilise the vital dye MTT. 3 μl ofstock MTT solution are added to each well, and then the plate isincubated at 37° C. for 1-2 hours during which time dehydrogenaseenzymes within the cells convert the soluble MTT to an insoluble blueformazan product that can be quantified spectrophotometrically.

In parallel, an 8-point standard curve is set up. Starting with thenumber of cells added to each upper chamber (100,000) and going down in2-fold serial dilutions in GBSS+BSA, the cells are added to a plate in25 μl, with 3 μl of MTT stock solution added. The standard curve plateis incubated along side the migration plate.

At the end of this incubation, the liquid is carefully removed from thelower chambers, taking care not to disturb the precipitated formazanproduct. After allowing to air dry briefly, 20 μl of DMSO is added toeach lower chamber to solubilise the blue dye, and absorbance at 595 nmis determined using a 96-well plate reader. The absorbance of each wellis then interpolated to the standard curve to estimate the number ofcells in each lower chamber.

The chemoattractant stimulated migration is determined by subtractingthe average number of cells that reached the lower compartment in wellswhere no chemoattractant was added from the average number of cells thatreached the lower compartment where the chemoattatractant was present.

The impact of the test substance is calculated by comparing thechemoattractant-induced migration which occurred in the presence orabsence of various concentrations of the test substance. Typically, theinhibition of migration is expressed as a percentage of the totalchemoattractant-induced migration which was blocked by the presence ofthe compound. For most compounds, a dose-response graph is constructedby determining the inhibition of chemoattractant-induced migration whichoccurs at a range of different compound concentrations (typicallyranging from 1 nM to 1 μM or higher in the case of poorly activecompounds). The inhibitory activity of each compound is then expressedas the concentration of compound required to reduce thechemoattractant-induced migration by 50% (the ED₅₀ concentration).Typically, MCP-1 induced migration of THP-1 cells has been used as thestandardised test system for the comparison of the biological activityof a wide range of compounds (see for example Reckless & GraingerBiochem J. 1999 Jun. 15; 340 (Pt 3):803-11; Reckless et al. Immunology.2001 June; 103(2):244-54; Fox et al. J Med Chem. 2002 Jan. 17;45(2):360-70; Fox et al. J Med Chem. 2005 Feb. 10; 48(3):867-74; theInternational applications supra). Compounds which inhibit leukocytemigration induced by more than one chmokine, but not by non-chemokinechemoattractants (such as TGF-β or C5a) are defined as Broad-spectrumChemokine Inhibitors (BSCIs; see for example Grainger & Reckless BiochemPharmacol. 2003 Apr. 1; 65(7):1027-34; Grainger et al. Mini Rev MedChem. 2005 September; 5(9):825-32).

Results

A typical dose response curve for compound (I′) inhibiting MCP-1 inducedmigration of THP-1 cells is shown in FIG. 6, together with comparabledose response curves for other selected lactam BSCIs known to haveparticularly high (that is, <1 nM) potency. The potency of compound (I′)versus various chemokines and non-chemokine chemoattractants, expressedas ED50 values is shown in Table 6, and is compared with other lactamBSCIs described previously.

It is clear from this data that compound (I′) can be classified as aBSCI (since it powerfully and potently inhibits leukocyte migrationinduced by a range of chemokines, but has no effect on leukocytemigration induced by a non-chemokine chemoattractant, in this case theC5a anaphylatoxin). Furthermore, it is evident that compound (I′) is atleast as potent and powerful as a BSCI in vitro as the selected lactamBSCIs which have previously been disclosed (for example, compound (II)in WO2006/016152 or compound (IV) in WO2005/053703). All of the lactamBSCIs examined here are considerably more potent than any of thenon-lactam BSCIs which have been disclosed to date (including imides,such as NR58,4, yohimbamides, lysergamides and peptide 3 and relatedstructures such as NR58-3.14.3). Indeed, compound (I′) is more potent asa BSCI in vitro (at least against MCP-1 induced migration) than anyother compound disclosed or described previously. Although this potencyas a BSCI is quantitatively superior to BSCIs in the prior art (albeitto a small degree), it is not this property that primarily marks outcompound (I) as unexpectedly superior to the prior art BSCIs. Instead,this demonstrates that the unexpected, and substantially superior, ADMEand pharmacokinetic properties of compound (I) compared to a wide rangeof previously disclosed BSCIs, has been achieved with no loss of poweror potency as a BSCI in vitro.

TABLE 6 Effect of selected lactam BSCIs on leukocyte migration in vitro.Com- IL-8 C5a pound MCP-1 SDF-1α RANTES neutrophils neutrophils (II) 80100 100 600 >1,000,000 (III) 80 Not tested Not tested Not tested Nottested (V) 120 200 250 500 >1,000,000 (I′) 50  50  80 600 >1,000,000(IV) 800 Not tested Not tested Not tested Not tested In each case thedose of compound (in pM) required to inhibit leukocyte migration inresponse to a maximal dose of the stated chemoattractant by 50% (theED50) is shown. Unless stated otherwise, the data is reported for theTHP-1 cell lines. For C5a induced migration none of the compounds testedinhibited neutrophil migration to any degree even at the highestconcentration tested (1 μM).

Example 6 Anti-Inflammatory Activity In Vivo

We have used the sub-lethal LPS-induced endotoxemia assay to demonstratethe generalised anti-inflammatory properties in vivo of previouslydisclosed BSCIs (see, for example, Fox et al. J Med Chem. 2002;45(2):360-70; Fox et al. J Med Chem. 2005; 48(3):867-74). In this assay,mice are given a non-specific pro-inflammatory challenge using bacterialendotoxin (LPS), and the extent of the systemic inflammatory response(measured by serum levels of the central pro-inflammatory cytokineTNF-α, which is essentially absent from the blood under normalconditions, but is rapidly elevated in response to a wide range ofinflammatory stimuli). We have selected this model (even though it isnot, itself, a particularly close model of any human inflammatorydisease condition, but because TNF-α is known to be important in verymany diseases (including rheumatoid arthritis, autoimmune disorders,Crohn's Disease, atherosclerosis, asthma and many more). Consequently,agents which suppress TNF-α production are already used clinically (e.g.Enbrel™ and other anti-TNF-α antibody products) to treat a wide range ofsuch diseases. Demonstration of TNF-α suppressive activity in this modelis therefore highly predictive of a clinically useful anti-inflammatoryeffect in a wide range of diseases.

Mice (in groups of 6) were pretreated with various doses of eachcompound, either by the subcutaneous route 30 mins prior to LPSchallenge, or by the oral route (via gavage) 60 mins prior to LPS. Themice were then challenged with an intraperitoneal injection of 750 μg ofbacterial LPS and sacrificed 3 hours later. Serum was prepared from aterminal bleed by cardiac puncture, and the concentration of TNF-α isdetermined by ELISA (R&D Systems). In each experiment, a group of 6 micereceive no LPS to act as a negative control, and a second group receiveonly LPS (with no candidate inhibitor). The level of TNF-α in serum fromthese animals, which received LPS without drug pre-treatment, isarbitrarily set to 100% (and is typically of the order of 6,000 pg/ml,compared with levels of <10 pg/ml among the negative control group). Wehave previously shown that the synthetic corticosteroid dexamethasone(itself a well known anti-inflammatory medicament active in a wide rangeof inflammatory diseases) inhibits LPS-induced TNF-α production by atleast 90% in this model, while thalidomide (another published inhibitorof TNF-α production, acting at the level of cellular TNF-α productionrather than as a leukocyte recruitment inhibitor like the BSCIsdescribed here) inhibits LPS-induced TNF-α production by about 60%.

The effect of compound (I′) at various doses, as well as other selectedlactam BSCIs, is shown in FIG. 7. As expected, the compound powerfullyinhibits LPS-induced TNF-α production whether the compound is deliveredvia the subcutaneous route (circles) or oral route (triangles). At dosesabove 1 μg/mouse, LPS-induced TNF-α levels were generally suppressed bymore than 90%, comparable to the effects of the corticosteroiddexamethasone.

The other lactam BSCIs tested also inhibited LPS-induced TNF-α in adose-dependent manner (FIG. 7), although the potency in vivo of (I′) wasgreater than any of the other compounds tested (and, indeed, greaterthan the potency of other lactam BSCIs previously disclosed elsewherewhich have been tested in this assay). This quantitative (albeit small)increase in potency is not the primary reason that we hereby claimcompound (I) as unexpectedly superior to the prior art BSCIs. Instead,this demonstrates that the unexpected, and substantially superior, ADMEand pharmacokinetic properties of compound (I) compared to a wide rangeof previously disclosed BSCIs, has been achieved with no loss of poweror potency as an anti-inflammatory agent in vivo. In addition, thesefindings clearly demonstrate the utility of (I) as an anti-inflammatoryagent in vivo, in a model of inflammation which indicates utility in awide variety of inflammatory diseases where increased TNF-α productionis a component of the pathogenic mechanism.

It is important to note that the hyperacute inflammation observed inthis model is particularly insensitive to the ADME and pharmacokineticproperties of the anti-inflammatory agents tested. Since the LPSstimulation is administered only 30 minutes after the drug, even agentswith very short plasma residence times (such as compounds (II) and(III)) remain present in plasma at sufficient concentrations to elicit apowerful anti-inflammatory effect. While such a test, therefore, doesnot emphasise the superiority of the claimed compounds over the priorart, it nevertheless demonstrates the utility of the compound.

The utility of the claimed compound is further demonstrated by studiesin an animal model of the human disease asthma (the hyperacuteinflammatory response observed in response to LPS exposure may not betypical of any particular human disease, although it is clearly a usefulmodel system of acute inflammation in general). In these studies,rodents (typically rats) are exposed to ovalbumin in accordance with thefollowing experimental design:

Adult Brown Norway rats (200-300 g body weight; n=10 per group) weresensitised by a single intraperitoneal injection of 0.1 mg Ovalbumin onday 0. Each rat then received an intratracheal challenge with a solutionof 1% ovalbumin (w/v) on day 8, and with 2% ovalbumin (w/v) on days 15,18 and 21. The animals were then sacrificed 3 hours after the finalchallenge on day 21. Note that ovalbumin (Sigma; purest available grade)was made endotoxin-free by passage over EndoTrap™ Red columns (purchasedfrom Cambrex; used in accordance with the manufacturer's instructions),and the endotoxin level was confirmed as <5 EU/mg protein using the LALassay (QCL-1000™; Cambrex; performed in accordance with themanufacturer's instructions; 1 mg of standard endotoxin contains˜900,000 EU/mg). This ensures that the lung inflammation responseresults from the allergic response to the ovalbumin protein, rather thanfrom unintended LPS stimulation which occurs even with the highestpurity grade commercial ovalbumin preparations, and therefore ensuresthe model more closely represents the underlying molecular pathology ofhuman asthma.

One group of mice (acting as a baseline control) received no ovalbuminchallenges, but were otherwise treated identically. A second group(positive control) received the challenges but no drug treatment. Athird group were treated identically, but received daily dosage withcompound (I′) at a dose of 0.3 mg/kg via oral gavage from day 8 untilday 21, with dosage being given 1 hr prior to any subsequent challengewith ovalbumin made on the same day. Compound (I′) was administered as asterile solution in endotoxin-free phosphate buffered saline. A fourthgroup received monteleukast (the active component of the commerciallyavailable asthma medication Singulair™) at 30 mg/kg via the oral route,in an identical treatment schedule to compound (I′).

On sacrifice, total lung leukocyate recruitment was assessed byperforming a broncheoalveolar lavage (BAL) using 4 lots of 3 ml sterilephosphate-buffered saline introduced through a tracheal cannula. Foreach animal, the BAL washes were combined, and the total cell populationcounted (using a haemocytometer). Additionally, the types of leukocytepresent were estimated using a flow cytometer in accordance withprocedures well known in the art.

The spleen was also removed from each mouse and placed in RPMI+10%FCS+antibiotics. The spleen was then pressed through fine-mesh (100 μm)nylon screens in a sterile sieve cup placed in a sterile petri dish toproduce single-cell suspensions. The resulting cell suspension was thencentrifuged (328 g; 5 mins) and washed in RPMI+10% FCS+antibiotics,before being resuspended in fresh media and counted using ahaemocytometer.

4×10⁶ total splenocytes (excluding RBCs) in total were cultured (37° C.;5% CO₂) in RPMI-10% FCS+antibiotics overnight in presence of 2 U/ml (10ng/ml) murine IL-2 in 4 wells of a 96 well plate (100 μl volume perwell/1×10⁶ cells/well) from each mouse. Approximately 24 hrs later, the4 wells were split into two groups of 2 wells: one group were leftuntreated, while the second group were stimulated with 500 ng/mlIonomycin and 50 ng/ml PMA for 4 hours at 37° C. During the last twohours of this incubation 10 μg/ml Brefeldin A (stock 1 mg/ml in EtOH)was added to one well from each set. Brefeldin A blocks proteintransport to golgi and therefore allows accumulation of proteins in ER.

The wells without Brefeldin A were incubated for a further 48 hours at37° C. At the end of the incubation, the cell suspensions werecentrifuged (328 g; 5 mins) and the supernatant was subjected to ELISAassays (R&D Systems; performed in accordance with the manufacturer'sinstructions) for murine IL-4 (a marker of Th2 cells) and murineinterferon-γ (IFN-γ; a marker of Th1 cells).

The wells with Brefeldin A were stained for intracellular IL-4 and IFN-gimmediately at the end of the four hour incubation as follows: cellswere stained with anti-CD4-FITC antibody (eBioscience Rat IgG2b, Cat.Code. 11-0041) for 30 mins on ice, then washed in Dulbecco's PBS andfixed with 2% paraformaldehyde (final concentration) in Dulbecco's PBSfor 20 mins. After fixation cells were made permeable with Dulbecco'sPBS/1% BSA/0.5% saponin (Sigma S7900) for 10 mins at room temperature.The cells from each well were then split into three separate FACS tubesand incubated with:

-   -   IFN-g-PE (eBioscience Rat IgG1, Cat. Code. 12-7311-82, 100 μg)        OR    -   I1-4-PE (eBioscience Rat IgG1, Cat. Code. 12-7041-82, 100 μg) OR    -   Isotype controls (a mixture of Rat IgG2b-FITC, eBioscience Cat.        Code 11-4031 and Rat IgG1-PE, eBioscience Cat. Code 12-4301)        for 30 mins at room temperature. Cells were then washed (twice        with PBS/BSA/saponin and then with PBS/BSA without saponin to        allow membrane closure) and resuspended in Dulbecco's PBS ready        for flow cytometry analysis.

Cells with specific staining for CD4 on the FITC channel (identifyingthem as T-helper cells) were analysed for the presence of specificstaining for either IL-4 or IFN-g on the PE channel. The ratio of CD4+cells staining positive for IFN-g to CD4+ cells staining positive forIL-4 was then reported as the Th1/Th2 ratio. Untreated Brown Norway ratshave a Th1/Th2 ratio of approximately 2.7 in the spleen (that is,approximately 2.7 times more CD4+ cells in the spleen are synthesisingINF-g as IL-4). Following sensitisation and repeated challenge withovalbumin, the ratio had fallen to less than 1.5 demonstrating themarked Th2 polarisation which accompanies asthmatic changes in bothrodents and humans (a lower Th1/Th2 ratio indicates relative Th2polarisation, while an increasing Th1/Th2 ratio indicates a relative Th1polarisation).

Daily dosing with both compound (I′) and the positive-control comparitorcompound monteleukast significantly reduced the number of leukocytes inthe BAL washes (70% reduction with compound (I′); p<0.01 Student'sunpaired t-test; FIG. 8). This unequivocally demonstrates that thecompound of the invention has a useful anti-inflammatory effect in amodel of human asthma, resulting from its ability to block leukocytemigration in response to chemokines. The magnitude of such an effect isat least comparable with that of commercially available medicamentsintended for the treatment of human asthma (such as Singular™), whilethe excellent pharmacokinetic and biodistribution parameters of compound(I′) are illustrated by the considerably increased potency compared tomonteleukast (a dose of 30 mg/kg of monteleukast is required to generatea similar reduction in BAL leukocytye counts compared to a dose of only0.3 mg/kg of compound (I′)).

Daily dosing with compound (I′), but not with the positive-controlcomparitor compound monteleukast, significantly reversed the Th2polarisation (FIG. 9), which is considered a major driver of asthmapathogenesis both in the ovalbumin-induced lung inflammation model usedhere, and in human asthma. Treatment with compound (I′) even at lowdoses such as 0.3 mg/kg via the oral route completely abolishes the Th2polarisation caused by chronic exposure to allergens such as ovalbumin,such that the Th1/Th2 balance in animals treated with compound (I′) isessentially indistinguishable from unchallenged Brown Norway rats.

It is interesting to note that other chronic inflammatory diseases, suchas atherosclerosis, are associated with a Th1 (as opposed to a Th2)polarisation. In both types of diseases, the imbalance in the T-helpercell cytokine profile has been described as a major pathogenic cause ofthe chronic inflammatory component of the disease. In models of adisease associated with a Th1 polarisation (such as in atherosclerosis)we have previously observed a marked shift towards Th2 on treatment withBSCIs, such as also observed with compound (I′). Mice with a homozygousdeletion of the gene encoding apoE (apoE−/− mice) develop severalvascular lipid lesions, even on a normal chow diet, and have a Th1/Th2ratio of approximately 8 (compared to 3.2 in the background C57Bl6wild-type strain). However, following treatment with BSCIs for a 3 monthperiod (from 12 weeks of age to 24 weeks of age, the period during whichmost of the lipid lesion development occurs) normalises the Th1/Th2ratio (and even causes a Th2 polarisation at very high doses in thismodel). Taken together with the data in the ovalbumin-induced lunginflammation model of asthma, we have demonstrated that BSCIs are ableto normalise or rebalance the T-helper cell cytokine production profileirrespective of whether the underlying pathogenic defect is a Th2polarisation (as in asthma) or a Th1 polarisation (as inatherosclerosis). We believe that BSCIs are currently the only agentsdescribed which have this “rebalancing” effect on the T-helper cellpopulation. These mechanistic insights further underpin (together withefficacy data in numerous animal models of different diseases with aninflammatory component) our claim that BSCIs, and in particular compound(I) claimed here (as a result of its unexpectedly superiorpharmacokinetic and biodistribution properties) are useful asmedicaments to treat an unusually broad range of conditions with aninflammatory component.

1. A method of treatment, amelioration or prophylaxis of the symptoms ofan inflammatory disease selected from asthma, allergic rhinitis orchronic obstructive pulmonary disease comprising: administering to apatient in need thereof a composition comprising an effectiveanti-inflammatory amount of a compound of formula (I):

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1wherein the composition comprises at least one pharmaceuticallyacceptable excipient and/or carrier.
 3. The method of claim 1 whereinthe composition is intravenously administered.
 4. The method of claim 1wherein the composition is orally administered.
 5. The method of claim 1wherein the inflammatory disease is asthma.
 6. The method of claim 1wherein the inflammatory disease is allergic rhinitis.
 7. The method ofclaim 1 wherein the inflammatory disease is chronic obstructivepulmonary disease.
 8. A method of treatment, amelioration or prophylaxisof the symptoms of an inflammatory disease selected from asthma,allergic rhinitis or chronic obstructive pulmonary disease comprising:administering to a patient in need thereof a composition comprising aneffective anti-inflammatory amount of a compound of formula (I′):

or a pharmaceutically acceptable salt thereof.
 9. The method of claim 8wherein the composition comprises at least one pharmaceuticallyacceptable excipient and/or carrier.
 10. The method of claim 8 whereinthe composition is intravenously administered.
 11. The method of claim 8wherein the composition is orally administered.
 12. The method of claim8 wherein the inflammatory disease is asthma.
 13. The method of claim 8wherein the inflammatory disease is allergic rhinitis.
 14. The method ofclaim 8 wherein the inflammatory disease is chronic obstructivepulmonary disease.